Most genetic material in a bacterium exists as giant DNA molecules which are present as the chromosome of the cell. A certain amount of the genetic material may also be present in the form of smaller, closed circular DNA molecules known as plasmids. The portion of the DNA molecule related to a specific hereditary trait is called a gene.
By techniques referred to as genetic engineering, it is possible to transfer a gene, which codes for the production of a specific protein, from one microorganism to another. The microorganism which receives the new genetic material is referred to as the host. Various workers have used these techniques to provide microorganisms which are superior producers of certain specific proteins such as enzymes.
It has been discovered that plasmids, which contain a series of genes linked together in the form of a circle, can be removed from the cells of one microorganism and inserted into the cells of another microorganism with comparative ease. Plasmids can also be used as vectors to carry new genetic material into a host organism. This is accomplished by first cutting the plasmid with an enzyme, known as a restriction endonuclease, that opens the circle of DNA. A fragment of foreign DNA, containing the desired gene, is inserted into the place where the DNA circle was cut. The circle is reformed by treatment with DNA ligase. The recombined plasmid, a new circular DNA molecule, contains the genes of the original plasmid plus the new gene from the piece of DNA which was inserted. This plasmid can be introduced into a host microorganism. The plasmid containing the new gene is then reproduced in the host microorganism and becomes part of its genetic material.
For a host microorganism to be suitable for use in genetic engineering, it must be capable of incorporating the new DNA. Furthermore, it must yield a viable microorganism which expresses the traits coded in the newly inserted gene. For the microorganism to produce useful quantities of protein, the microorganism must also be one that can be grown on a commercial scale.
Experimenters using the new recombinant DNA technology have been concerned that the microorganisms with new genetic material might produce substances harmful to man, animals or plants. Because of this concern, the National Institute of Health (NIH) issued "Guidelines for Research Involving Recombinant DNA Molecules" in 1978. These guidelines provided for various levels of physical containment in laboratories where genetic engineering experiments are conducted. They also established levels of biological containment for microorganisms containing recombinant DNA.
Biological containment relates to the use of host cells and vectors which have limited ability to survive if they escape from the laboratories into the the natural environment. The NIH guidelines establish levels of biological containment for host-vector systems (hereafter designated HV) depending on the microorganisms and the genetic material used. HV1 is defined as a "host-vector system which provides a moderate level of containment". A high level of biological containment is required for a HV2 host-vector system.
The NIH has certified two mutants of B. subtilis for use as host components of HV1 systems. One of these, RUB331, is disclosed in U.S. Pat. No. 4,302,544, the disclosure of which is incorporated herein by reference in its entirety. The second of these is BGSC No. IS53 which is described by Ellis, D. M. and Dean, D. H., Recombinant DNA Technical Bulletin, Vol. 4, No. 1, March, 1981. The NIH has approved one B. subtilis host component for a HV2 system. This strain, known as ASB298, is described by Burke, W. F. and Le, H. T., Recombinant DNA Technical Bulletin, Vol. 3, No. 1, December, 1980.
The present invention describes a new asporogenous mutant designated as B. subtilis EE101 (ATCC 39,096) useful as a host in a host-vector system. This host has the additional advantage of being an amylase-negative mutant. Such a mutant is particularly suitable for use as a host for recombinant plasmids which contain genes coding for the production of specific amylases such as thermostable alpha-amylases. These amylases are of commercial importance for use in processes to make starch hydrolyzates, glucose, and high fructose syrups. Since the mutant does not require an antibiotic for growth, it is a more practical host for enzyme production than the approved HV2 host, ASB298, which grows only in the presence of streptomycin.